disruption of the genes encoding antigen 85a and antigen 85b of

INFECTION AND IMMUNITY,0019-9567/00/$04.0010

Feb. 2000, p. 767–778 Vol. 68, No. 2

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Disruption of the Genes Encoding Antigen 85A and Antigen 85B ofMycobacterium tuberculosis H37Rv: Effect on Growth

in Culture and in MacrophagesLISA Y. ARMITIGE,* CHINNASWAMY JAGANNATH, AUDREY R. WANGER,

AND STEVEN J. NORRIS

Department of Pathology and Laboratory Medicine, University of Texasat Houston Medical School, Houston, Texas

Received 15 October 1999/Accepted 9 November 1999

The mechanism of pathogenesis of Mycobacterium tuberculosis is thought to be multifactorial. Among theputative virulence factors is the antigen 85 (Ag85) complex. This family of exported fibronectin-binding pro-teins consists of members Ag85A, Ag85B, and Ag85C and is most prominently represented by 85A and 85B.These proteins have recently been shown to possess mycolyl transferase activity and likely play a role in cellwall synthesis. The purpose of this study was to generate strains of M. tuberculosis deficient in expression of theprincipal members of this complex in order to determine their role in the pathogenesis of M. tuberculosis.Constructs of fbpA and fbpB disrupted with the kanamycin resistance marker VKm and containing varyingamounts of flanking gene and plasmid vector sequences were then introduced as linear fragments into H37Rvby electroporation. Southern blot and PCR analyses revealed disruption of the homologous gene locus in onefbpA::VKm transformant and one fbpB::VKm transformant. The fbpA::VKm mutant, LAa1, resulted from adouble-crossover integration event, whereas the fbpB::VKm variant, LAb1, was the product of a single-cross-over type event that resulted in insertion of both VKm and plasmid sequences. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis and Western blot analysis confirmed that expression of the disrupted gene wasnot detectable in the fbpA and fbpB mutants. Analysis of growth rates demonstrated that the fbpB mutant LAb1grew at a rate similar to that of the wild-type parent in enriched and nutrient-poor laboratory media as wellas in human (THP-1) and mouse (J774.1A) macrophage-like cell lines. The fbpA mutant LAa1 grew similarlyto the parent H37Rv in enriched laboratory media but exhibited little or no growth in nutrient-poor media andmacrophage-like cell lines. The targeted disruption of two genes encoding mycolyl transferase and fibronectin-binding activities in M. tuberculosis will permit the systematic determination of their roles in the physiology andpathogenesis of this organism.

It has been estimated that one-third of the world’s popula-tion is infected with Mycobacterium tuberculosis, the causativeagent of the disease tuberculosis (24). The incidence of tuber-culosis continues to increase worldwide, particularly amonggroups such as the medically underserved, the immunosup-pressed, and the confined (11). New concerns have been raisedby the reported increase in the overall number of cases, as wellas by the increase in number of drug-resistant isolates (10, 11).Investigations into potential drug targets, more-efficient vac-cines, and more-effective treatment regimens are currently un-der way in an effort to decrease morbidity and mortality due tothis disease. While numerous immunogenic antigens and pu-tative virulence factors have been isolated, cloned, and se-quenced (36), insight into the mechanisms of pathogenesis ofM. tuberculosis remains elusive.

The antigen 85 (Ag85) complex is a family of fibronectin-binding proteins that are considered to be potential virulencefactors. These proteins (Ag85A, Ag85B, and Ag85C, encodedby the genes fbpA, fbpB, and fbpC, respectively) garnered at-tention when M. tuberculosis was found to bind selectively tofibronectin and not to other purified extracellular matrix pro-teins tested (32). Binding of these organisms to purified fibro-nectin is dose dependent and can be blocked by antibodies

produced either to fibronectin or to members of the Ag85complex (31, 32). It has been clearly demonstrated that theability to bind fibronectin and other extracellular matrix pro-teins enhances the virulence of pathogenic organisms, mostnotably staphylococci and streptococci (28). Specific binding tohost extracellular matrix proteins may aid in the adherence anddissemination of organisms in tissue.

Members of the Ag85 complex are both secreted and re-tained in the cell wall of M. tuberculosis (1). Quantitatively, theproteins are secreted at a ratio of 3:2:1, Ag85A to Ag85B toAg85C (15). These proteins have recently been found to pos-sess mycolyl transferase activity (9), adding to the growingnumber of cell wall-synthetic enzymes identified in mycobac-teria (3, 4, 6, 10). Belisle et al. (9) identified members of theAg85 complex as enzymes responsible for the transfer of my-colic acids to a-a9-trehalose to form a-a9-trehalose monomy-colate (TMM) and a-a9-trehalose dimycolate (TDM), alsoknown as cord factor. Ag85A and Ag85C share a similar spe-cific mycolyl transferase activity, while the specific activity ofAg85B is only about 20% of that of Ag85C. One recent study(19) has shown that a disruption mutant of a clinical isolate ofM. tuberculosis deficient in Ag85C contained 40% less cellwall-bound mycolates than the parent strain. These mycolatesrepresented not only TMM and TDM but also other moleculessuch as arabinogalactan, glycerol monomycolate, a-mycolates,methoxymycolates, and ketomycolates. There was no apparentchange in the composition of mycolates detected, but theirquantity was affected by mutation of Ag85C. It remains to be

* Corresponding author. Mailing address: Department of Pathologyand Laboratory Medicine, 6431 Fannin St., Houston, TX 77030. Phone:(713) 500-5272. Fax: (713) 500-0730. E-mail: [email protected].

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determined why mycobacteria possess three enzymes with ap-parently similar activities and whether the individual membersof the Ag85 complex play different roles in the production ofthese molecules.

In this study, several constructs were utilized in an attemptto achieve homologous recombination and disruptional mu-tagenesis of fbpA and fbpB in the virulent M. tuberculosis strainH37Rv. PCR, Southern blot hybridization, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), andimmunoblot analyses confirmed the disruption and inactiva-tion of fbpA and fbpB in individual strains of M. tuberculosis.Loss of FbpA expression was shown to inhibit the ability ofH37Rv to grow in wholly synthetic media or to replicate inhuman or mouse macrophage-like cell lines, indicating thatFbpA may play a role in the pathogenesis of M. tuberculosis.

MATERIALS AND METHODS

Bacterial strains and media. Middlebrook media were purchased from DifcoLaboratories, Detroit, Mich. The virulent laboratory strain of M. tuberculosis,H37Rv (ATCC 27294), was grown in liquid Middlebrook 7H9 medium supple-mented with 0.2% glycerol, 0.25% Tween 80 (Sigma Chemical Co., St. Louis,Mo.), and albumin-dextrose complex (ADC) (consisting of 0.5% bovine serumalbumin, fraction V [Sigma], 0.085% NaCl, and 0.2% glucose) or Sauton medium(25). Middlebrook 7H10 or 7H11 plates supplemented with ADC with or without20 mg of kanamycin/ml were used for colony isolation. Cycloheximide at 50 mg/mlwas added to all plates to inhibit growth of fungi during incubation. Escherichiacoli DH5a1 (Stratagene, La Jolla, Calif.) was used for recombinant DNA studiesand plasmid propagation. These strains were grown on solid or liquid Luria-Bertani (LB) medium (34) supplemented with 50 mg of kanamycin/ml as indi-cated.

Macrophage cell lines and media. The human monocyte-like cell line THP-1(ATCC TIB-202) and the murine monocyte-like cell line J774A1 (ATCC TIB-67) were maintained in nitrate-free RPMI 1640 medium (GIBCO BRL, GrandIsland, N.Y.) supplemented with 50 mg of HEPES/liter, 200 mM glutamine, 2.2 gof sodium bicarbonate/liter, 50 mg of L-arginine/liter, 100 U of penicillin/ml, 50mg of gentamicin/ml, and either 10% heat-inactivated human AB serum (forTHP-1 cells) or 10% heat-inactivated fetal bovine serum (FBS) (for J774A1cells). Cells for use in M. tuberculosis cocultures were expanded in media withoutantibiotics and harvested during log phase.

Recombinant DNA techniques. To isolate chromosomal DNA, M. tuberculosiscells were grown to confluence on Lowenstein-Jensen slants at 37°C under 9.5%CO2. Cells were scraped from the surface of the slant and resuspended in 500 mlof TE buffer (10 mM Tris–1 mM EDTA [pH 8]) in a 1.5-ml microcentrifuge tube.Two milligrams of achromopeptidase (Sigma) per milliliter was added, and thesamples were incubated for 1 h at 37°C. At that time, 120 mg of proteinase K/mland 1.5% SDS were added, and samples were incubated for 10 min at 65°C.N-cetyl-N,N,N-trimethyl ammonium bromide (1.3%, vol/vol) was added, and thesamples were incubated for another 10 min at 65°C. These samples were thenextracted twice with phenol-chloroform (1:1) and once with chloroform-isoamylalcohol (24:1) and were ethanol precipitated. DNA was pelleted and resus-pended in an appropriate volume of TE buffer. Molecular cloning and restrictionendonuclease digestion were performed by standard techniques (34). Cloningvectors used were pBluescript KS(1) (Stratagene) and pNEB193 (New EnglandBiolabs, Beverly, Mass.). Restriction endonucleases and other enzymes (NewEngland Biolabs; Promega, Madison, Wis.) were used according to the manu-facturers’ instructions. The VKm cassette in plasmid pHP45VKm (14) was gra-ciously provided by the laboratory of Malcolm Winkler, Department of Micro-biology and Molecular Genetics, University of Texas—Houston Medical School.

Generation of transforming plasmids. A 9.4-kb BamHI/ClaI fragment con-taining the H37Rv fbpA gene was cloned into pBluescript KS by standard meth-ods (34) and identified by Southern blot hybridization using a PCR-generatedfragment of fbpA as a probe. An internal 750-bp ApaI fragment was subclonedinto pBluescript KS(1), excised by using the vector KpnI and EcoRI sites, andthen ligated into pNEB193 linearized with the same two enzymes. The resultantplasmid, pLYAa6, was linearized at the unique SacII site within fbpA (see Fig. 1)and treated with the Klenow fragment of DNA polymerase I to provide bluntends. A blunt-ended VKm cassette was prepared by liberating the VKm frag-ment from pHP45VKm with EcoRI and treating the resultant fragment with theKlenow fragment. The blunt-ended pLYAa6 and VKm DNA fragments wereligated to generate plasmid pLYAa6VKm, containing an fbpA::VKm gene dis-ruption (see Fig. 1). An internal 500-bp AccI fragment was subcloned directlyinto the AccI site of pNEB193 and likewise interrupted with the VKm cassette atthe SacII site to generate the transforming plasmid pLYAa4VKm (see Fig. 1).

For fbpB::VKm gene disruptions, a 6.6-kb EcoRI/HindIII fragment containingthe fbpB gene of H37Rv was cloned into the EcoRI and HindIII sites of pBlue-script KS. An internal 750-bp SacII fragment was subcloned into pBluescript KSand then into pNEB193 by using the vector SacI and XbaI sites to generate

pLYAb5. This plasmid was linearized with EcoRV and ligated with a blunt-endedVKm cassette, prepared as described above, to generate plasmid pLYAb5VKm(Fig. 1). A 2.3-kb HindIII/NcoI fragment containing fbpB was subcloned intopNEB193 to generate pLYAb3. This plasmid was mutated by addition of ablunt-ended VKm cassette at the unique EcoRV site within fbpB to generatepLYAb3VKm (Fig. 1).

To prepare the DNA for transformation of M. tuberculosis, plasmidspLYAa4VKm and pLYAb5VKm were linearized with SacI and pLYAa6VKmwas linearized with ClaI (Fig. 1). In addition, pLYAa6VKm and pLYAb5VKmwere treated with ApaI and SacII, respectively, to yield the mutated geneswithout vector sequences.

The shuttle plasmid pLYAspk was generated by cloning the 3-kb KpnI/EcoRVorigin of replication fragment of the Mycobacterium fortuitum plasmid pAL5000from the recombinant plasmid pYUB18 (20) into pBluescript KS and cloning theVKm marker into the unique BamHI site. This plasmid is able to replicate inboth E. coli and M. tuberculosis and to confer kanamycin resistance.

Electroporation of M. tuberculosis H37Rv. M. tuberculosis H37Rv was preparedfor electroporation as previously described (20). Briefly, cells were grown inMiddlebrook 7H9 medium–ADC–Tween 80 with gentle shaking to an opticaldensity at 600 nm of 0.6 to 1.0, washed three times in 1/50 volume of cold 10%glycerol, resuspended in 10% glycerol at a concentration of ;1011 cells/ml, andstored at 270°C until needed. The linearized plasmids (2 to 4 mg of DNA)diagrammed in Fig. 1 were electroporated at 0°C into 1010 electrocompetentH37Rv cells by using an Electroporator 2510 (Eppendorf North America, Mad-ison, Wis.) at a setting of 1,250 V; under these conditions, the pulse time was 4to 5 ms. One milliliter of 7H9–ADC broth without antibiotics was added imme-diately, and the bacteria were incubated at 37°C for 2.5 h with agitation. Trans-formants were then plated on 7H10–ADC–kanamycin plates and incubated at37°C for 3 weeks under 9.3% CO2. Individual kanamycin-resistant colonies weresubcultured onto fresh 7H10–ADC–kanamycin plates and grown an additional 2to 3 weeks prior to further evaluation.

Hybridization analysis. Fluorescein conjugation of the DNA fragments usedas probes and chemiluminescent detection of hybridization were carried outaccording to the GeneImages procedure (Amersham Life Science Inc., ArlingtonHeights, Ill.). For initial screening of transformants, chromosomal DNA liber-ated from boiled M. tuberculosis colonies was immobilized onto a Hybond N1nylon membrane (Amersham Life Science Inc.) by using a slot blot apparatus.Immobilized DNA was hybridized with a fluorescein-labeled 700-bp fragmentfrom the VKm cassette obtained by digestion with PvuII, agarose gel separation,and purification using the PCR Cleanup kit (Promega). In addition, intact chro-mosomal DNA was digested with the enzymes indicated in Fig. 3, electropho-resed in 0.7% agarose, and transferred to a nylon membrane by using thealkaline transfer procedure (34). The membrane was then hybridized with afluorescein-labeled probe (either the internal 750-bp ApaI fragment from fbpA orthe internal 750-bp SacII fragment from fbpB).

PCR. Primers used in this study are listed in Table 1. PCR was performed toscreen for disruptions in fbpA (see Fig. 2) and fbpB (data not shown). Twoprimers were unique to the upstream regions of fbpA (59A2) and fbpB (59B2),and one primer was common to both fbpA and fbpB (39AB). The primers 59VKmand 39VKm were used to screen for the presence of the VKm cassette. Cells (104

to 106 per reaction) from transformants were boiled in TE buffer for 10 min toliberate chromosomal DNA, which was used as a template. PCR conditions were10 mM Tris (pH 8.8), 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 mMdeoxynucleoside triphosphates, 0.5 mM each primer, and 2 U of Thermalasepolymerase (Amresco, Solon, Ohio) per 100 ml of reaction mixture. Denatur-ation, annealing, and extension temperatures (and times) were as follows: 1 cycleof 96°C for 2 min; 5 cycles of 94°C for 40 s, 56°C for 40 s, and 72°C for 1.5 min;30 cycles of 94°C for 40 s, 68°C for 40 s, and 72°C for 1.5 min; and a finalextension of 72°C for 10 min.

Sequence analysis. The PCR primer pairs 59A2 and 39VKm2 and 59VKm2 and39Aexp (see Fig. 2) were used to amplify the 59 and 39 regions, respectively, of thefbpA gene locus in LAa1. Primers 39VKm2 and 59VKm2 are specific for se-quences within the VKm cassette. PCR primers 59B2 and 39VKm2 were used toamplify the 59 region of the fbpB locus of LAb1. Southern blot analysis revealed

TABLE 1. PCR primers used for analysis of fbpA and fbpB mutants

Primer Sequence

59A2 ..................................59-TTGATGCGGGTGGACGCC-3939Aexp................................59-GATCAAGCTTGTTCGGAGCTAGGCGCCC-3959B2 ..................................59-GCGGCTGTAGTCCTTCC-3939Bexp ................................59-GATCAAGCTTTCAGCCGGCGCCTAACG-3939AB .................................59-CGGCAGCTCGCTGGTCAGGA-3959VKm..............................59-TCTCACCTTGCTCCTGCC-3939VKm..............................59-GGTCCGCCACACCCAGCC-3959VKm2............................59-GAAGTAATCGCAACATCCGC-3939VKm2............................59-CAAGGGCTCCAAGGATCGGG-39pNEB1..............................59-GGTGTTGGCGGGTGTCGGGG-39

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that the transforming vector pNEB193 had integrated into the fbpB locus. PrimerpNEB1, which is specific for pNEB193, was used with 39Bexp to amplify the 39region of LAb1. PCR products were purified, desalted, and used directly astemplates for sequencing. DNA sequencing was performed by using an ABI 377automatic DNA sequencer (Perkin-Elmer/Applied Biosystems, Foster City, Cal-if.) at the DNA Core Laboratory, Department of Microbiology and MolecularGenetics, University of Texas–Houston Medical School. Sequences were ana-lyzed with the GAP and BESTFIT programs (Genetics Computer Group, Mad-ison, Wis.).

SDS-PAGE and Western blot analysis. The parent strain, H37Rv, and thefbpA::VKm and fbpB::VKm mutants were grown in stationary cultures (withoutagitation) in Middlebrook 7H9 broth without additional supplements, except 20mg of kanamycin/ml for selection of the two mutant strains. Five milliliters of11-day cultures were filtered twice through a 22-mm-pore-size filter. Two milli-liters of each filtrate was concentrated to ;150 ml by using a Centricon-10membrane apparatus (Amicon, Beverly, Mass.). The retentates were resus-pended in an equal volume of solubilization buffer (2% SDS, 5% 2-mercapto-ethanol, 10% glycerol). Proteins were electrophoresed in 8-to-20% polyacryl-

FIG. 1. Plasmids generated for transformation of M. tuberculosis H37Rv. Open boxes, fbpA and fbpB open reading frames; hatched boxes, signal sequences; heavylines, vector sequences. Numbers in parentheses are distances between restriction sites. (A) fbpA::VKm constructs. A 500-bp AccI fragment and a 750-bp ApaI fragmentwere cloned from fbpA into pNEB193, a ColE1 plasmid, and mutated by the addition of an VKm cassette at the SacII site. Plasmid pLYAa4VKm was digested withSacI and introduced into H37Rv as a linear fragment, while pLYAa6VKm was electroporated into H37Rv associated with (by digestion with ClaI) and liberated from(by digestion with ApaI) the vector pNEB193. (B) fbpB::VKm constructs. A 750-bp ApaI fragment and a 2.3-kb HindIII/NcoI fragment were cloned from fbpB intopNEB193 and mutated by the addition of an VKm cassette at the EcoRV site. Plasmid pLYAb5VKm was introduced into H37Rv associated with (by SacI digestion)and liberated from (by SacII digestion) the vector pNEB193.

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amide gradient gels and stained with Coomassie blue or transferred to polyvinyldifluoride (PVDF) membranes as previously described (27). Western blot anal-ysis was carried out by incubating PVDF membranes with a 1:100 dilution ofhybridoma culture supernatant containing the monoclonal antibody HYT27 (1),kindly provided by T. M. Shinnick, Centers for Disease Control and Prevention,Atlanta, Ga. This antibody has been shown to react with FbpA, FbpB, and FbpC.Second antibody incubation and detection were carried out according to theGeneImages procedure (Amersham).

In vitro growth studies. The parent strain, H37Rv, and the fbpA and fbpBmutants were washed with saline and added at 104 CFU/ml to the enrichedMiddlebrook 7H9–ADC and synthetic Sauton media and incubated with gentlemixing at 37°C for 14 days. On days 3, 5, 7, 10, and 14, aliquots were removedfrom each broth culture and plated in 10-fold dilutions on 7H10–ADC plateswith or without kanamycin for colony counts.

Infection of macrophages. Macrophage infections were carried out as previ-ously described (21, 22). Briefly, THP-1 cells were washed three times in theRPMI 1640 assay medium described above but with 2% AB serum, 1 mg oftetrahydrobiopterin/ml, and no antibiotics. Suspensions of M. tuberculosis H37Rvor the fbpA or fbpB mutant were dispersed by gentle sonication and mixed with108 THP-1 cells at a concentration of 1010 CFU in 5 ml of assay medium.Phagocytosis was allowed to occur for 4 h with gentle mixing at 37°C. Cells werethen washed by low-speed centrifugation and resuspension in assay medium sixtimes, diluted to 106 cells/ml, and plated at 1 ml per well in 24-well culture plates.For infection of J774A1 cell cultures, adherent monolayers were established in24-well plates by using RPMI 1640 assay medium with 10% FBS. Monolayerswere infected at a CFU/macrophage ratio of 1:1 for 4 h with gentle mixing at37°C. The monolayers were then washed extensively with warm assay medium.Twenty-four-well plates with infected THP-1 and J774A1 cells were incubated at37°C under 5% CO2, and 1 ml of fresh assay medium was added to each well onday 3. Aliquots of THP-1 cells and supernatants were aspirated from wells,pelleted, and lysed with 0.05% SDS in saline, while J774A1 cells were lysed insitu on days 0 (baseline CFU), 3, 5, and 7 (three replicates per time point). SDSlysates were neutralized by the addition of sterile 15% bovine serum albumin insaline, and lysates were diluted in sterile saline. Serial 10-fold dilutions of thediluents were plated out on 7H11 agar for CFU counts. Controls for extracellulargrowth of M. tuberculosis were obtained by incubating 104 CFU of M. tuberculosisH37Rv/ml in 1 ml of assay medium containing a sonicated lysate of 106 THP-1macrophages which had been passed through a 0.22-mm-pore-size filter to re-move cell debris. Heat-inactivated human AB serum does not support M. tuber-culosis growth, nor does M. tuberculosis grow in RPMI 1640 assay mediumcontaining macrophage lysate and 5% heat-inactivated AB serum (data notshown).

Electron microscopic examination of M. tuberculosis-infected THP-1 cells. Onday 7 post-macrophage infection, aliquots of THP-1 cells infected with H37Rv orLAa1 were aspirated from wells, pelleted, and washed with phosphate-bufferedsaline three times. The cells were then prepared for electron microscopy aspreviously described (16).

RESULTS

Transformation of M. tuberculosis. H37Rv was transformedby electroporation with 2 to 4 mg of several linearized con-structs containing the fbpA and fbpB genes interrupted by theVKm cassette (Fig. 1). The transforming fragments containedbetween 74 and 585 bp of the M. tuberculosis sequences oneither side of the VKm insert; in some cases the vector se-quences were retained to protect the construct from potentialexonuclease activity. These constructs lacked an origin of rep-lication active in M. tuberculosis; therefore, kanamycin resis-tance could be conferred only if part or all of the transformingfragment was integrated into the M. tuberculosis chromosome.Plasmid pLYAspk, which contains the VKm cassette and is

capable of replication in both M. tuberculosis and E. coli, wasused as a positive control for transformation. Following elec-troporation, the transformation mixtures (originally containing;1010 M. tuberculosis cells) were plated on 7H10–ADC–kana-mycin plates to select for kanamycin-resistant (Kmr) colonies.To assess the rate of Kmr due to spontaneous mutation versusintegration or recombination events, transformants were sub-jected to slot blot analysis using a 700-bp fragment of VKm asthe probe.

The results obtained are summarized in Table 2. The trans-formation efficiency, calculated by transformation with the au-tologously replicating plasmid, pLYAspk, was ;105 transfor-mants/mg of DNA. In this experiment, 57 to 389 Kmr colonieswere obtained in the transformations utilizing fbpA::VKm andfbpB::VKm constructs. However, 17 Kmr colonies were identi-fied in the negative control without transforming DNA, indicat-ing the occurrence of spontaneous mutations leading to Kmr.Indeed, only a small proportion of the Kmr colonies represent-ing organisms transformed with fbpA::VKm or fbpB::VKmconstructs contained VKm, as determined by slot blot analysis(Table 2). Two constructs, pLYAa6VKm and pLYAb5VKm,integrated into the chromosome in 3 of 10 (30%) and 9 of 26(34.6%) of the Kmr colonies screened (Table 2). Recombi-nants containing the VKm cassettes were not detected in thelimited number of clones examined from the other transfor-mation groups.

PCR screening. To identify H37Rv clones with the desiredfbpA::VKm and fbpB::VKm gene disruptions, a PCR strategywas devised to amplify the region where the targeted disrup-tion was to occur. Each PCR mixture contained a primerunique to the upstream region of fbpA (59A2), a primer uniqueto the upstream region of fbpB (59B2), and a primer that wascommon to both (39AB) (Fig. 2A). Under the PCR amplifica-tion conditions used, an integrative or recombinative event atone or the other gene locus would result in a correspondingloss of product. The amplification of both genes thus providedan internal PCR control.

Ninety-six Kmr colonies each from the fbpA::VKm andfbpB::VKm transformations were screened in this manner. Theresults of several of the fbpA::VKm transformants are shown inFig. 2A. Most of the amplification reactions resulted in anintact fbpA product (600 bp) and an intact fbpB product (300bp). Transformants that contained the fbpB PCR product butlacked the fbpA PCR product were chosen for further analysis.Additional primers specific for VKm and different regions offbpA (Fig. 2B) were used to further characterize the mutants byPCR. One fbpA::VKm transformant was found to have a dis-ruption in the 59 region of the gene, while the 39 region wasintact. By using primers to VKm sequences, this transformantwas found to contain the VKm cassette (Fig. 2B, rightmostpanel). The VKm cassette was used as a positive control in thiscase. Four fbpB::VKm transformants were analyzed in a simi-

TABLE 2. Results obtained from electroporation of M. tuberculosis H37Rv with different fbpA::Km and fbpB::Km constructs

Transforming DNA (restrictionenzyme used for linearization)

Gene construct(6 vector sequences)

No. of Kmr

coloniesNo. of colonies with

VKm/total no. screenedDisruption mutants

identified

No DNA 17 Not donepLYAa4VKm (SacI) fbpA::VKm (1 vector) 68 0/10pLYAa6VKm (ClaI) fbpA::VKm (1 vector) 53 3/10 1pLYAa6VKm (ApaI) fbpA::VKm (2 vector) 254 0/14pLYAb5VKm (SacI) fbpB::VKm (1 vector) 57 9/26 1pLYAb5VKm (SacII) fbpB::VKm (2 vector) 389 0/30pLYAa4VKm (SspI) fbpB::VKm (1 vector) 366 0/28pLYAspk (Control plasmid) 9.09 3 104 1/1



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lar manner, and one was found by PCR to contain a 59 disrup-tion of fbpB, an intact 39 region, and VKm sequences (data notshown). Screening was discontinued when single fbpA::VKmand fbpB::VKm mutants were obtained. These two disruptionmutants were analyzed further.

Southern blot analysis of fbpA::VKm and fbpB::VKm mu-tants. Digestion of wild-type H37Rv chromosomal DNA withthe restriction endonuclease ApaI generates a 750-bp fbpAgene fragment (Fig. 1), while digestion with EcoRI generates a5-kb fragment with the 1,059-bp fbpA gene located near the

FIG. 2. PCR analysis of M. tuberculosis H37Rv transformed with constructs pLYAa4VKm treated with SacI (a4-SacI), pLYAa6VKm treated with ClaI (a6-ClaI),and pLYAa6VKm treated with ApaI (a6-ApaI). (A) Initial screening strategy. The locations of the primers used are indicated in the diagram. DNA from pLYAa4VKm-and pLYAa6VKm-transformed Kmr colonies was used as a template in a PCR using three primers: one unique to the upstream region of fbpA (59A2), one unique tothe upstream region of fbpB (59B2), and one common to both fbpA and fbpB (39AB). The PCR conditions used allowed complete amplification of ;1 kb of DNAtemplate (the product for fbpA is 600 bp and that for fbpB is 300 bp), but not of larger products with VKm inserts. Several transformants had a 300-bp PCR productrepresenting fbpB but no 600-bp product representing intact fbpA. (B) PCR verification and characterization of fbpA::VKm mutants. The locations of the primers usedare shown in the diagram. A single pLYAa6VKm transformant (starred; lane a6-ClaI) lacked a wild-type 59 fbpA (59A2339AB) (left panel), while its 39 region wasintact (middle panels). This clone was also found to possess the VKm cassette used for selection (right panel). The fbpB::VKm transformants were screened in anidentical fashion, and a single fbpB::VKm transformant with a single-crossover insertion was identified (data not shown).



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center (12). When chromosomal DNA from the fbpA::VKmmutant was digested with ApaI and probed with a PCR-gen-erated fbpA fragment, it was found to lack the 750-bp nativegene fragment. Instead, it now contained a 3-kb fragment iden-tical in size to the transforming DNA fragment (Fig. 3A).Digestion of DNA from this transformant with EcoRI revealedthat the locus had undergone an increase in size roughly equiv-alent to the size of the VKm cassette (;2.3 kb). In addition,EcoRI-digested DNA from the transformant was probed withthe labeled transforming vector, pNEB193, and vector se-quences were found to be absent (Fig. 3C). These data areconsistent with the occurrence of a double-crossover recombi-nation event at the fbpA locus.

The fbpB gene locus in the fbpB::VKm mutant was evaluatedin a similar manner. Digestion of H37Rv chromosomal DNAwith the restriction endonuclease SacII generates an fbpB genefragment of approximately 750 bp, and digestion with KpnIgenerates a ;7-kb fragment containing fbpB. Analysis of thefbpB::VKm mutant by digestion with SacII and KpnI andSouthern blot hybridization with the labeled 750-bp SacII fbpBgene fragment revealed the presence of the native gene frag-ment in addition to the transforming fragment (Fig. 3B). Thisresult is consistent with an integrative event in which bothfbpB::VKm and vector sequences were inserted at the nativefbpB site. This interpretation was corroborated by the fact thatthe 7-kb KpnI fragment increased to ;12 kb in the fbpB::VKmmutant. Thus, the pLYAb5VKm fragment was inserted at thenative gene locus through a single-crossover event near the 59end, as supported by the presence of vector sequences at thissite (Fig. 3C). The fbpA::VKm and fbpB::VKm mutants weredesignated LAa1 and LAb1, respectively.

Sequence analysis. Sequence analysis was performed onLAa1 and LAb1 to characterize the recombination events thathad occurred. Analysis of the 59 and 39 regions of the fbpAgene sequences flanking the VKm cassette in LAa1 revealedthat the sequences were identical to those of the wild-type genein these regions (data not shown). These results were con-sistent with a double-crossover, homologous-recombinationevent within the fbpA gene (Fig. 4A). Sequence analysis of the59 region of LAb1 also revealed sequence identity to wild-typefbpB in this region. The sequence downstream of the VKmcassette was identical to the end of the construct pLYAb5VKm(including the segment of pNEB193), indicating that this re-gion was integrated in its entirety into the fbpB::VKm genelocus. Surprisingly, the 39 end of the inserted sequence termi-nated at the SacI site used for linearization of the transformingfragment (Fig. 4C); therefore, no degradation of the end of theinsert occurred before integration. Following the insert se-quence, the wild-type fbpB gene resumed at bp 107 of the openreading frame, marking the exact point of insertion. A modelof the integrative event occurring in LAb1 is shown in Fig. 4B.

SDS-PAGE and Western blot analysis. To verify that thechromosomal mutations in LAa1 and LAb1 disrupted synthe-sis of FbpA and FbpB, respectively, concentrated supernatantproteins and whole-cell lysates from wild-type H37Rv and thetwo mutants were analyzed by SDS-PAGE. Results for super-natant and lysate proteins were the same. As demonstrated inFig. 5A, the parent, H37Rv, had two prominent protein bandsat 32 kDa (representing FbpA) and 31 kDa (representingFbpB). The FbpA band was absent in LAa1, while the othervisible protein bands were all intact. Likewise, the protein bandrepresenting FbpB was absent in LAb1. Western blot analy-

FIG. 3. Southern blot analysis of H37Rv and fbpA::VKm and fbpB::VKm transformants. In each panel, open arrows indicate the locations of hybridizing bandscorresponding to wild-type H37Rv sequences, whereas solid arrows indicate bands containing the VKm disruption. (A) Chromosomal digest of H37Rv and the fbpA::VKm transformant LAa1, which has a disruption of the 59 region of fbpA. The probe is the 750-bp ApaI fragment of fbpA. (B) Chromosomal digest of H37Rv and thefbpB::VKm transformant LAb1 with a 59-region disruption of fbpB by PCR analysis. The probe is the 750-bp SacII fragment of fbpB (Fig. 1). The transformant possessesboth the wild-type SacII fragment and the fbpB::VKm fragment. (C) Chromosomal digest of fbpA::VKm and fbpB::VKm transformants hybridized with the vectorpNEB193. LAa1 lacks the vector, while LAb1 retains the vector. Additional bands observed in some lanes are due to cross-hybridization of the fbpA or fbpB probe withother fbp genes.



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sis with monoclonal antibody HYT27 confirmed these results(Fig. 5B). HYT27 is reactive with all members of the Ag85complex and bound specifically to the FbpA and FbpB bandsin H37Rv, the FbpB band only in LAa1, and the FbpA band inLAb1. The quantity of FbpC was apparently too low in thesepreparations to be detected by these techniques.

In vitro growth studies. Since the Ag85 complex has beenidentified as having mycolyl transferase activity, the fbpA andfbpB mutants were anticipated to display differences during invitro culture. In order to test this hypothesis, the parent andmutant strains were cultured in enriched Middlebrook 7H9–ADC broth or Sauton broth, a commonly used synthetic me-dium. While all three strains grew in the Middlebrook 7H9–

ADC broth at very similar rates, the fbpA mutant, LAa1, wasunable to grow in the synthetic Sauton medium (Fig. 6).

Macrophage infection studies. The macrophage cell line cul-ture method can be used to measure differences in capacityfor intracellular growth among strains of mycobacteria (21,22). Human THP-1 macrophage cultures were infected withH37Rv and with the fbpA and fbpB mutants LAa1 and LAb1to determine intracellular growth. In this system, the parentstrain, H37Rv, increased in number from 104 to $106 myco-bacteria per culture over a 7-day incubation period (Fig. 7).These results are similar to those obtained with M. tuberculosisErdman in the same system (22). The fbpB mutant, LAb1,exhibited a similar growth curve. However, the fbpA mutant,

FIG. 4. Results of the analysis of the M. tuberculosis H37Rv transformants LAa1 and LAb1B. (A) Model of the fbpA mutant, LAa1; (B) model of the fbpB mutant,LAb1; (C) sequences from the 39 “joint” region of the fbpB::VKm mutant, LAb1. Comparisons to the transforming vector sequence (pNEB193) and the wild-type fbpBsequence are shown. Only one difference of 1 bp (corresponding to bp 108 in the fbpB coding region) was observed in the joint region.



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LAa1, actually decreased in number under the same incuba-tion conditions, indicating an inability to survive and replicatein macrophages (Fig. 7B). Monolayers of the J774A1 murinemacrophage cell line also supported the growth of the parent

strain, H37Rv, and the fbpB mutant, whereas the fbpA mutantdid not replicate (Fig. 7A). Examination by electron micros-copy (Fig. 8A) revealed that infection of THP-1 cells by H37Rvresulted in high numbers of intact, intracellular mycobacteria.In contrast, LAa1 infection resulted in fewer mycobacteria thatwere rarely intact and exhibited varying degrees of degrada-tion. LAa1-infected cells also had many extensions indicativeof membrane ruffling and cell activation (Fig. 8B), whereasH37Rv-infected cells appeared relatively quiescent.

DISCUSSION

While homologous recombination with gene replacementhas been demonstrated readily in fast-growing, nonpathogenicmycobacteria, such as Mycobacterium smegmatis (17), genera-tion of defined mutations in the genomes of slow-growingmycobacteria initially proved difficult. Attempts to achieve ho-mologous recombination in M. tuberculosis and Mycobacteriumbovis BCG revealed a high rate of illegitimate recombination,with random insertion of linear DNA fragments into the chro-mosome (23, 35). Despite this finding, intraplasmic (8, 26)and interplasmic (8) recombination experiments have demon-strated that homologous recombination does occur in slow-growing mycobacteria, although at a significantly lower ratethan illegitimate recombination. Aldovini et al. (2) obtainedrecombination without gene replacement at the chromosomaluraA site of M. bovis BCG, producing transformants that hadundergone single-crossover homologous recombination at onlyone end of the transforming fragment. These results indicatedthat disruption could be achieved by using fragments internalto the coding region of a gene. Other studies involving the useof linear DNA fragments of single-gene length (4, 33), linearDNA fragments of 40 to 50 kb (5), transposon delivery systems(7, 29, 30), and SacB counterselection plasmids (3, 30) haveproven successful with increasing frequency. Despite the in-creased proficiency at mutagenesis of the M. tuberculosis chro-

FIG. 5. SDS-PAGE and Western blot analysis of supernatant proteins fromH37Rv, LAa1, and LAb1. (A) Coomassie blue-stained SDS-PAGE gel of con-centrated supernatant proteins from H37Rv, LAa1, and LAb1 revealed thatLAa1 lacks the FbpA protein band and LAb1 lacks the FbpB protein band. Bothprotein bands were present in the parent, H37Rv. (B) Western blot analysis ofsupernatant proteins detected with monoclonal antibody HYT27.

FIG. 6. Effect of medium composition on the growth of fbpA- and fbpB-deficient M. tuberculosis mutants. Saline-washed M. tuberculosis H37Rv or an fbpA or fbpBmutant was inoculated into enriched liquid Middlebrook 7H9 medium or wholly synthetic Sauton medium. All strains grew similarly in the enriched Middlebrook7H9–ADC medium. The fbpA mutant, LAa1, was unable to grow in the Sauton medium, which lacks the ADC supplement.



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mosome, the list of identified potential virulence factors re-mains brief.

In this study, several constructs were utilized in our attemptsto disrupt the fbpA and fbpB chromosomal loci in M. tubercu-losis (Fig. 1). The size of the M. tuberculosis DNA insert rangedfrom 0.5 to 2.3 kb, and the amount of M. tuberculosis DNAflanking the VKm cassette ranged from 74 bp to 1.3 kb. Linearconstructs with and without flanking vector sequences wereused. Each of the six constructs tested was modeled after priorattempts to disrupt genes in M. tuberculosis (2, 23, 26). Wereasoned that using several constructs would increase the like-lihood of gene disruption.

Insertional mutants were obtained for both fbpA and fbpB.Of the six constructs utilized for transformation in this study,only two yielded H37Rv clones containing VKm cassettes (Ta-ble 2). Both of these (pLYAa6VKm, treated with ClaI, andpLYAb5VKm, treated with SacI) had relatively small M. tu-berculosis DNA inserts (;750 bp) and contained flanking vec-tor DNA. Although the number of clones examined was toosmall for statistical analysis, we can conclude that insertionalmutagenesis of M. tuberculosis genes can be achieved withsmall inserts and may be aided by the presence of flankingvector sequences. Our efforts were focused on the single fbpA::VKm and fbpB::VKm mutants described here, but it is likelythat additional site-specific mutations could be identified withadditional screening.

In the case of fbpA, mutagenesis occurred by double-cross-over homologous recombination, resulting in an insertion ofVKm into the chromosomal fbpA site. This result was con-firmed by PCR, Southern blot, and sequence analyses. PCRresults using primers within fbpA and VKm and primers out-side the region used for transformation were consistent with adouble-crossover event without addition of extraneous DNA.By Southern blot analysis, there is an increase in size at thenative fbpA gene locus equal to the size of the VKm cassette.Sequence data confirmed addition of the VKm cassette at theSacII site of fbpA without addition or deletion of base pairs.With regard to fbpB, Southern blot analysis revealed that theentire transforming fragment, including the vector pNEB193,

had integrated into the chromosome. Sequencing of this regiondemonstrated the occurrence of homologous recombination atthe 59 end of the gene with a double-stranded break andinsertion of the nonhomologous vector sequence adjacent tothe fbpB chromosomal sequences at the 39 end.

Results of previous attempts to mutate individual genes inslow-growing mycobacteria using chromosomal fragments mu-tated with a kanamycin resistance marker have suggested apredominance of illegitimate over legitimate recombination.This was based on the presence of a high number of antibiotic-resistant colonies in the absence of the desired gene disruption.Our studies suggest that there is a high rate of spontaneouskanamycin resistance in these organisms and that a large num-ber of the Kmr colonies reported previously were due to spon-taneous mutation rather than illegitimate recombination. Ofthe 118 Kmr colonies that we screened (Table 2), 106 (90%)were spontaneous kanamycin mutants, in that slot blot analysisrevealed the absence of the kanamycin resistance marker. Ofthe remaining 12 transformants that were found to have inte-grated the VKm cassette into their chromosomes, only 2 (17%)had undergone integration at the homologous gene loci. Pre-vious studies have involved screening of transformants for thedesired mutation by looking for a phenotypic change (i.e.,auxotrophy or urease activity) and have not addressed the rateof spontaneous mutation. We have found that the rate ofspontaneous mutation is somehow increased by the electropo-ration of DNA but not by the act of electroporation itself(unpublished data). As an example, the rate of spontaneousmutation was lower under control conditions where no DNAwas used during electroporation (Table 2).

Members of the Ag85 complex have been found to possessmycolyl transferase activity. Since disruption of members ofthe complex could potentially affect cell wall synthesis, weexamined the abilities of the two mutants, LAa1 and LAb1, togrow in routine laboratory media, one ADC-containing me-dium and one minimal medium composed of basic salts with nosupplements. There was no significant difference in the growthrates of the mutant strains compared to that of the parentstrain in media containing ADC (Fig. 6A). In contrast, the

FIG. 7. Growth of H37Rv and the fbpA and fbpB mutants in monocyte-like human THP-1 and murine J774A1 cell cultures. J774A1 (A) or THP-1 (B) cells wereinfected with the parental H37Rv strain, the fbpA mutant LAa1, or the fbpB mutant LAb1 for 4 h and plated at 106 cells/culture, as described in Materials and Methods.On days 0, 3, 5, and 7, the cell cultures were harvested, lysed, and plated for mycobacterial CFU counts on 7H11 agar with appropriate antibiotics. Mean CFU 6standard deviations for representative experiments are shown.



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FIG. 8. Poor survival and growth of the fbpA disruption mutant LAa1 in THP-1 cells, as revealed by electron microscopy. THP-1 cells were inoculated with eitherthe wild-type progenitor, H37Rv (A), or LAa1 (B) and were processed for electron microscopy 7 days postinfection. The H37Rv-infected cells contained many intact,electron-dense mycobacteria (solid arrows), whereas LAa1-infected cells contained few intact mycobacteria and many vacuoles containing apparent mycobacterial celldebris (open arrows).



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fbpA mutant, LAa1, exhibited little growth in a minimal me-dium that lacked ADC (Fig. 6B). Albumin-containing enrich-ments are added to the growth media of mycobacteria primar-ily to bind toxic lipid byproducts produced during routinegrowth in the presence of lipids such as Tween 80. The albu-min-oleate complex also acts as an additional nutrient sourcefor the organisms. The lack of growth of the fbpA mutant inminimal medium may indicate an increased dependence onlipids or other compounds associated with albumin in the en-riched medium. In macrophage-like cell line infection modelswith both human and mouse cell lines, disruption of Ag85Bagain had no obvious effect on growth compared to that of theparent strain while the Ag85A mutant, LAa1, was severelyhampered in its growth (Fig. 7). The number of CFU of LAa1actually decreased during macrophage cell line infection, andkilling of the mycobacteria by macrophages was verified byelectron microscopy (Fig. 8A). Poor survival of LAa1 in mac-rophages may reflect an alteration in phagosome processingsuch that the mycobacteria are exposed to lysosomal contentsor phagosome acidification (13). Alternatively, the increaseddependence on nutritional compounds observed in broth cul-tures could result in decreased survival and growth in theintracellular compartment. These possibilities will be ad-dressed in subsequent studies.

It has been postulated that the members of the Ag85 com-plex, though closely related, are not coordinately regulated(15). In keeping with this hypothesis, we found no evidence inour mutants that expression of the other genes of the complexincreased or decreased quantitatively in response to the loss ofone of the members (Fig. 5). The same study also demon-strated that the genes are transcribed as monocistronic mes-sages, decreasing the likelihood that the effects seen in thisstudy are due to a polar effect on genes downstream of thefbpA gene locus.

Much speculation has been made about the roles of theindividual members of the Ag85 complex. Although they allhave mycolyl transferase activity, there is clear evidence thatthe efficiency at which this reaction is carried out differs amongthe three proteins (9). Studies have shown that naked DNAvectors containing the Ag85A gene injected into mice affordeda promising degree of protection as a vaccine against M. tu-berculosis infection (18), which would seem to indicate a rolefor the Ag85 complex in the immunology of this process. Mu-tation of individual members of this complex will aid in deter-mining the individual roles of these proteins in the pathogen-esis and immunology of mycobacterial infection as well as inthe synthesis of the mycobacterial cell wall.

ACKNOWLEDGMENTS

We thank T. M. Shinnick, Centers for Disease Control and Preven-tion, for providing monoclonal antibodies, M. E. Winkler, Departmentof Microbiology and Molecular Genetics, University of Texas—Hous-ton Medical School, for supplying plasmid pHP45VKm, and W. R.Jacobs, Department of Microbiology and Immunology, HowardHughes Medical Institute, Albert Einstein College of Medicine, Bronx,N.Y., for providing plasmid pYUB18. We also thank G. M. Weinstock,H. B. Kaplan, E. M. Walker, and S. Mueller for invaluable advice anddiscussions, Betty Boulet and Emem Akpaffiong for technical assis-tance, and Patricia Navarro for electron microscopy.

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